Utilization of Spent Coffee Grounds as Media for Stone Pine (Pinus pinea) Seedlings

ORIGINAL PAPER

Utilization of Spent Coffee Grounds as Media for Stone Pine (

Pinus

pinea) Seedlings

Servet Caliskan1&Nihan Ozok2&Ender Makineci3 Received: 3 April 2020 / Accepted: 21 May 2020

# Sociedad Chilena de la Ciencia del Suelo 2020 Abstract

The present study was aimed at determining the influence of adding different amounts of spent coffee grounds (SCG) on germination percentage (GP) and mean germination time (MGT), seedling characteristics, and seedling biomass of different components of containerized 1 + 0 stone pine seedlings. Six different media were prepared including 100% conventional media, 100% sand, 10% SCG + 90% sand, 20% SCG + 80% sand, 30% SCG + 70%, sand and 100% SCG. While GP is 90% in the media where the SCG ratio is 10%, GP values decrease to 50% as the SCG ratio increases. The greatest seedling height values were found in conventional media (14.4 cm), followed by the seedlings in 100% sand with 11.4 cm and 20% SCG + 80% sand with 11.1 cm. The highest root collar diameter was found in 20% SCG + 80% sand with 3.9 mm. The highest root/shoot ratio (0.7) was obtained in 10% SCG + 90% sand. Total seedling biomass was the greatest in conventional media (2.1 g). As SCG increased in the media, N, K, Mg, and P concentrations increased, and Ca concentration and C/N ratio decreased in all seedling components (needle, main root, lateral root, and stem). Results point to the possibility of using a small amount of SCG to alter seed germination, seedling characteristics, and nutrient uptake for production of containerized stone pine seedlings.

Keywords Biomass . Fertilizer . Organic amendment . Sustainable . Waste

1 Introduction

Coffee is globally the second most valuable trade commodity after petroleum. It is among the most consumed beverages around the world. Everyday, approximately 3.5 billion cups of coffee are consumed worldwide. Production of coffee gen-erates a lot of coffee wastes and by-products that could be

used for various applications (Blinová et al.2017). Spent cof-fee grounds (SCG) are the waste item from cofcof-fee brewing. A ton of coffee beans produces 650 kg of SCG as a result of coffee preparation in coffee shops (Murthy and Madhava Naidu2012). As coffee consumption rises around the world, a significant amount of organic matter emerges from the prep-aration stage up to the stage of drinking, and this amount increases from year to year. As a result, more than two million tons of organic coffee waste, such as grounds, roasted crust, or ground coffee waste, emerges for one million tons of coffee production per year (Pandey et al.2000). Therefore, strategies for low-cost and effective recycling of coffee wastes have become very important (Yamane et al.2014).

In containerized seedling production, growth media may consist of a single material, but usually a mixture of several materials is used. Soil can be mixed with one or several media: sand, peat, leaf mold, perlite, fertilizer, etc. in order to prepare a good compound for germination, rooting, and growing. Soil fertilization aims to provide essential nutrients to plants so that they can develop normally, fruit optimally, and to improve the physical, biological, and chemical characteristics of the soil to a desirable extent for plants (Ciesielczuk et al.2015). Like * Servet Caliskan servetc@istanbul.edu.tr Nihan Ozok nihanozok@hotmail.com Ender Makineci emak@istanbul.edu.tr 1

Faculty of Forestry, Silviculture Department, Istanbul

University-Cerrahpasa, Bahcekoy, Sariyer, 34473 Istanbul, Turkey

2

Istanbul University-Cerrahpasa, Institute of Graduate Studies, Avcılar, 34325 Istanbul, Turkey

3 _{Faculty of Forestry, Soil Science and Ecology Department, Istanbul}

University-Cerrahpasa, Bahcekoy, Sariyer, 34473 Istanbul, Turkey

https://doi.org/10.1007/s42729-020-00271-5

many organic wastes, SCG is also used in plant growing me-dia. SCG influences plant growth by improving soil proper-ties. It increases water holding capacity, the amount of avail-able water to the plant, porosity volume, reduces soil bulk density, and can form aggregates in the soil (Cervera-Mata et al.2019a). Due to its high nutrient content, SCG also im-proves plant nutrition. It contains various minerals, of which potassium is the most abundant element, followed by phos-phorus and magnesium (Mussatto et al.2011).

Coffee wastes also contain high amounts of organic compounds (fatty acids, lignin, cellulose, hemicellulose, and other polysaccharides). There have been studies in connection with production of bio-diesel (Caetano et al. 2012), sugar (Mussatto et al. 2011), activated carbon (Kante et al.2012; Pappa et al.2012; Reffas et al.2010; Tsai et al.2012), compost (Preethu et al. 2007), and re-tention of metal ions (Fiol et al. 2008; Oliveira et al. 2008).

SCG has been used in many trials for plant growth. Hardgrove and Livesley (2016) tried using SCG directly with broccoli, leek, radish, viola, and sunflower. Cervera-Mata et al. (2019b) investigated plant nutrient concentra-tions in lettuces grown on agricultural soil by adding SCG directly. Other researchers have tested the use of SCG with other additives. For instance, Ronga et al. (2016) investigated a mixture of peat with SCG up to 40%, while Cruz et al. (2015) investigated the effect of 10% SCG and soil mixture on Batavia lettuce plant growth. Ribeiro et al. (2017) examined the application of ash and SCG mixture with Lolium perenne grass.

Nearly 0.7 million hectares of stone pine-dominated forests exist around the Mediterranean basin (Mutke et al.2012). Stone pine has been utilized for various pur-poses including environmental restoration, watershed and soil conservation, dune stabilization, and afforestation in-side urban areas (Boydak and Çalışkan 2014, 2015; Çalışkan and Boydak2017; Mutke et al.2012). In addi-tion to these diverse purposes, it is mostly known for its edible kernels that have been consumed by humans since Paleolithic times because of their high nutrition value (Evaristo et al.2010).

All studies on the effects of adding of SCG on plant growth involve plant species used in agricultural products. There are no studies conducted with woody species.

The aim of the present study was to determine the influence of the addition of different amounts of spent coffee grounds on: (1) seed germination, (2) seedling traits (height, main root length, maximum root length, root collar diameter, number of lateral branches), (3) biomass of seedling components (needles, stems, main root, and lateral roots), and (4) elemen-tal concentrations (N, C, Ca, Mg, P, and C/N) of seedling components of the containerized 1 + 0 stone pine seedlings in outdoor nursery conditions.

2 Material and Methods

2.1 Site Description

The trial was carried out in outdoor nursery conditions at the Forest Nursery Directorate in Bahçeköy/Sarıyer/ Istanbul. The nursery is located at a height of 126 m above sea level at the northern latitude of 41° 10′ 56″ and the eastern longitude of 28° 59′ 14″. The climate around the nursery is a humid, mesothermal, and maritime climate with a moderate deficit of water in the summer months, according to Thornthwaite’s classification. Mean annual precipitation is about 1111.4 mm, and mean annu-al temperature is 12.8 °C. Most of the precipitation occurs between the months of October and March (Akburak et al. 2018).

2.2 Seed Material, Preparation of Media and Sowing

of Stone Pine Seeds

Three-year-old stone pinecones were collected from 20 ma-ture trees in Bergama, Izmir-Turkey, making sure that there was a minimum distance of 75 m between them. Seeds ex-tracted from cones were floated to identify filled ones. The filled seeds were placed in plastic bags and stored in a refrig-erator at an approximate temperature of 3 °C ± 1 °C until sowing time in the nursery.

Plastic containers (18 cm tall and 190 cm−3) were used for sowing the seeds. For the trial, a randomized block design with four replications was used with six different media for each replication. The SCG used in nursery trials was obtained from different brewing methods of grounded Robusta and Arabica beans. The SCG collected over 3 months was stored and was dried in 105 °C prior to use. By volume, these media included 100% conventional media, 100% sand, 10% SCG + 90% sand, 20% SCG + 80% sand, 30% SCG + 70% sand, and 100% SCG. The media used conventionally in nurseries and the media formed with 100% sand were used as control material for the investigation of the effect of SCG used in different proportions on the germination and development of stone pine seeds. The conventional media in the nursery con-tains two units of soil, one unit of peat, and one unit of river sand. The properties of the media used are given in Table 1. The stone pine seedlings were irrigated twice a day, early in the morning and late in the evening. No fertilizers were applied to stone pine seedlings. As the SCG ratio increased, C, N, Mg, P, K, and salinity values rose significantly (Table 1). At the beginning of March, 1080 sound stone pine seeds were planted in the nursery with one seed in each container. Germinations were assessed two or three times a week for 120 days. GP

was calculated as the ratio between the number of germi-nated and non-germigermi-nated seeds.

MGT =∑(t × n)/∑n

where t represents the number of days from the test start, while n represents the number of germinated seeds on the day t (Bewley and Black1994).

2.3 Element Analysis of Samples

Using a LECO Truspec CN-2000 analyzer, all media samples were analyzed by means of dry combustion for their C and N concentrations. Exchangeable cations (K, Ca, Mg, Na, Mn) as 5 g samples were extracted with a standard neutral (pH≈ 7) 1 N ammonium acetate solution at 1:5 extracting ratio (w/v; soil to solution), then shaken for 20 to 30 min, and then fil-tered. ICP/OES was used to determine the concentrations of individual cations. A microprocessor pH-meter was used to determine acidity and an electrical conductivity (EC) meter for electrical conductivity (Akburak et al.2018).

Biomass component samples were dried at 70 °C until a constant weight is achieved. Sub-samples (0.5 g) were obtain-ed from the ground samples, placobtain-ed in Teflon tubes after which 4 ml concentrated HNO3(nitric acid) and 2 ml H2O2 (hydrogen peroxide) were added, and then samples were con-verted into solution in microwave digestion systems. Solutions were then prepared with ultra-pure water until the final volume reached 50 ml and were stored at 4 °C until analyzed. ICP/OES was used to determine the P, K, Ca, and Mg content and concentrations of the solutions (Çakır and Akburak 2017). Also, C and N concentrations of biomass components were analyzed with LECO Truspec CN-2000 analyzer.

2.4 Biomass Samplings

Twelve seedlings were harvested from each media, and each one was divided into its components of needles, stems, main root, and lateral roots. For dry weight determination, the sub-samples of seedling components were dried at 70 °C. Seedling height, main root length, maximum root length, root collar diameter, number of lateral branches, total needle weight, stem weight, main root weight, and lateral root weight were determined. Above-ground biomass, below-ground biomass, and total seedling biomass were determined, and then the dry weights of different seedling components were proportioned to total seedling biomass.

2.5 Statistical Analysis

The mean values and standard deviations were utilized to represent the seedling traits. To evaluate the differences be-tween treatments, analysis of variance (ANOVA) was con-ducted, and comparison of means was analyzed by

Table 1 Chemical composition o f spent coffee g round (S CG) added m edias Me di a N (% ) C (%) C/ N Ca (m g k g − 1) K (m g k g − 1) Mg (m g k g − 1) P (m g k g − 1) pH EC (μSc m − 1) Conventio nal m edia 0.22b (0.01) 3.81c (0.09) 17.08d (0.08) 4027.64f (36.88) 356.5 7 e (2.49) 905.39d (0 .81) 34.17d (0 .54) 6.58c (0.08) 535.67c (1 5.0 6) 100% Sand 0.16a (0.01) 1.60a (0.06) 9.78ab (0.19) 1988.42d (13.24) 66.02 a (0.42) 122.06a (0. 76) 1.16a (0.28) 7.02d (0.07) 132.5a (3.72) 10% SCG + 90% Sand 0.22b (0.01) 2.08a (0.10) 9.45a (0.15) 1214.90a (1 5.95) 85.37 b (0.86) 127.06a (0.56) 9.20b (1.34) 6.68c (0.03) 323.33b (2.19) 20% SCG + 80% Sand 0.24b (0.004 ) 2 .46ab (0.11) 10.30bc (0 .27) 1895.57c (2 4. 19) 185.9 1 c (2.47) 207.00b (1 .67) 27.13c (0.62) 6.35b (0.07) z697.67d (19. 72) 30% SCG + 70% Sand 0.29c (0.01) 3.16bc (0.04) 10. 84c (0.14) 1383.78b (12.11) 345.5 8 d (0.56) 272.48c (3 .49) 50.08e (1.74) 5.66a (0.10) 932.00e (6 5.90) 100% SCG 2 .00d (0.003 ) 33.43d (0.76) 16,70 (0.3 7) 2662.04e (1 1.89) 629. 9 4f (1.29) 2836.19e (18.32) 884.32f (4.8 7) 7.09d (0.03) 2336.67f (86.47) P 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 Values are the mean, numbers in parent hesis are st andard error, and v alu es in the same column fo llow ed b y the same le tt er ar e not sta tist ica lly diff er ent at the 0 .05 significance lev el in the D uncan pos t hoc te st

Duncan’s multiple range tests. Differences were considered significant when p < 0.05. SPSS software was used for all statistical analysis (SPSS2010).

3 Results

Seeds germinated between 86 and 94%, respectively, in conventional and 100% sand media, which were deter-mined as control material (Table2) (Fig.1). In the media where the SCG content was 10% (as in conventional and 100% sand media), the seeds germinated very successful-ly at 90% and the GP began to decline gradualsuccessful-ly due to the proportional increase in SCG, while GP values de-creased to 50% in the media created with 100% SCG. GP values were higher and MGT values were lower than conventional media GP values in the media mixed with 10% SCG (Table2, Fig. 1).

One-year-old seedlings generally comprised different characteristics of components in various SCG-containing

media (Fig. 2). The highest values in seedling height, maximum root length, total needle weight, stem weight, main root weight, lateral root weight, total above-ground biomass, below-above-ground biomass, and total bio-mass were found in seedlings grown in conventional media (Table 3). The longest main root length was de-termined in 100% sand media. Thus, in terms of these properties, the addition of SCG to a media had a reduc-ing effect. However, the greatest root collar diameter and number of lateral branches were found in 20% SCG + 80% sand media. The greatest root/shoot, lateral root weight/total seedling biomass, and below-ground biomass/total seedling biomass were determined in 10% SCG + 90% sand media. The media containing 100% SGC had the smallest values in all the mentioned parameters (except main root length), while it had the greatest values in the parameters of needle weight/total seedling biomass, stem weight/total seedling biomass, main root weight/total seedling biomass, and above-ground biomass/total seedling biomass (Table 3).

Fig. 1 Influence of spent coffee grounds (SCG) added media on the germination of stone pine (Pinus pinea) seeds

Table 2 Germination percentages (GP) and mean germination times (MGT) of stone pine (Pinus pinea) seeds in the various media with the different rates of spent coffee grounds (SCG)

Conventional media 100% Sand 10% SCG + 90% Sand 20% SCG + 80% Sand 30% SCG + 70% Sand 100% SCG P

GP 86 (2.74)ac 94 (1.75)a 90 (3.14)ac 77(6.86)ac 59 (13.60)bc 51 (10.82)b 0.025

MGT 49 (2.60)ac 42 (0.48)b 46 (0.91)bc 50 (1.49)ac 55 (1.44)a 52 (2.87)ac 0.002

Values are the mean, numbers in parenthesis are standard error, and values in the same row followed by the same letter are not statistically different at the 0.05 significance level in the Duncan post hoc test

Concentrations of N, C, Ca, K, Mg, P, and C/N in different components of stone pine seedlings are pre-sented in Table 4. The N concentration increased in all seedling components due to the increase in SCG rates in the growing media. C concentrations in different biomass components of seedlings were about 50%. The main root C concentrations of stone pine seedlings were not statistically different under different growing media. The amount of Ca in the seedlings decreased due to the increase in SCG ratio in growing media. Increasing the

SCG rate by 10, 20, and 30% raised K values in dif-ferent components of seedlings compared with the me-dia containing 100% sand. No significant Mg tendency due to SCG increase was observed in different seedling components. Parallel to the increase in SCG added to the growing media, a significant rise in P concentrations was observed in seedling needles, main roots, and stems (except lateral roots). As the amount of SGC in growing media increased, the C/N ratio in the tissues of seed-lings decreased (Table 4).

Fig. 2 Influence of spent coffee grounds (SCG) added media on the growth of stone pine (Pinus pinea) seedlings

Table 3 Influence o f spen t coffee grounds (SCG) added m edia on the stone p ine (Pinus pinea ) se edl ing tra its Conventional m edia 100% S and 10% S C G + 90% S and 20% SCG + 80% Sand 30% SCG + 70% Sand 100% SC G P Seedling height (cm) 14.43 (0.54 ) a 11.35 (0.44) c 10.85 (0.52) c 11.09 (0.35) c 10.27 (0.55) c 7 .23 (0.47) b 0 .0 00 Main root length (cm) 15.69 (0.62 ) ab 17.70 (1.26) a 15.58 (0 .29) ab 14.43 (0.30) b 13.85 (0.50) b 13.92 (1.24) b 0 .0 06 Maximum root length (cm) 26.76 (1.13 ) a 23.44 (1.43) ac 24.73 (2.23) ac 22.98 (1.06) ac 21.86 (0.91) c 16.80 (1.26) b 0 .0 01 Root collar d iameter (mm) 3 .72 (0.09) a 3 .59 (0.13) a 3 .6 7 (0.14) a 3 .87 (0.15) a 3 .52 (0.22) a 2 .56 (0.30) b 0 .0 00 Nu mber of lateral b ranches 7 .83 (0.37) cd 6.75 (0.33) c 8 .2 5 (0.41) ad 9.25 (0.60) a 8 .17 (0.39) ad 4.44 (0.56) b 0 .0 00 Total needle w eight (g) 1.04 (0.06) a 0.77 (0.04) c 0.76 (0.06) c 0.79 (0.05) c 0.75 (0.04) c 0.27 (0.05) b 0.0 00 Stem weight (g) 0.34 (0.03) a 0.26 (0.02) d 0.20 (0.02) c 0.21 (0.01) c 0.18 (0.01) c 0.1 (0.02) b 0.0 00 Main root weight (g) 0 .25 (0.02) a 0 .22 (0.02) ad 0.19 (0 .01) cd 0.18 (0.01) cd 0.17 (0.02) c 0.1 (0.02) b 0.0 00 Lateral root weight (g) 0 .49 (0.03) a 0 .47 (0.03) ac 0.47 (0.03) ac 0.40 (0.03) c 0 .38 (0.03) c 0 .05 (0.01) b 0 .0 00 Ab ove-ground biomas s (g) 1.39 (0.08) a 1 .03 (0.06) c 0 .9 6 (0.07) c 0 .99 (0.06) c 0 .93 (0.05) c 0 .37 (0.06) b 0 .0 00 Below-gro und biomass (g) 0.74 (0.04) a 0 .69 (0.04) ad 0.66 (0.03) acd 0.58 (0.03) cd 0.55 (0.04) c 0.15 (0.02) b 0.0 00 Total seedling b iomass (g) 2 .13 (0.11) a 1 .72 (0.10) c 1 .62 (0.10) c 1 .57 (0.09) c 1 .49 (0.08) c 0 .52 (0.08) b 0 .0 00 Root/shoot 0.54 (0.02) c 0.67 (0.02) a 0.70 (0.03) a 0 .59 (0.03) c 0 .59 (0.03) c 0 .41 (0.04) b 0 .0 00 Needle weight/total seedling biomass 0 .49 (0.01) c 0 .45 (0. 01) b 0 .47 (0.01) b 0 .50 (0.01) c 0 .51 (0.01) ac 0.53 (0.01) a 0 .0 00 Stem weight/total seedling b iomass 0.16 (0.005 ) c 0.15 (0. 005) c 0 .12 (0.004) b 0 .13 (0.004) b 0 .12 (0.01) b 0 .18 (0.01) a 0 .0 00 Main root weight/total seedling biomass 0 .12 (0.003 ) b 0.13 (0 .01) b 0 .12 (0.01) b 0 .12 (0.01) b 0 .12 (0.01) b 0 .19 (0.01) a 0 .0 00 Lateral root weight/total seedling b iomass 0.23 (0.01) c 0 .27 (0.01) ad 0.29 (0.01) a 0 .2 5 (0.01) cd 0.25 (0.01) cd 0.10 (0.02) b 0 .0 00 Ab ove-ground Bio m ass/total seedling b iomass 0.65 (0.01) d 0 .60 (0.01) bc 0.59 (0.01) b 0 .6 3 (0.01) cd 0.63 (0.01) cd 0.71 (0.02) a 0 .0 00 Be low-gro und B ioma ss/t o ta l seed ling b iomass 0.35 (0.01) c 0 .40 (0.01) ad 0.41 (0.01) a 0 .3 7 (0.01) cd 0.37 (0.01) cd 0.29 (0.02) b 0 .0 00 Values are the mean, numbers in parenthes is are st andard error, and v alues in the same row followed b y the sa me le tt er ar e not st at isti cal ly dif fe re n t at the 0.05 significance level in the Duncan post hoc test

Table 4 Elemental concentrations of st one pin e (Pinus pinea ) seedlings o n spent coffee grou nd (SCG) added m ed ia C onventional M edia 100% Sand 10% S C G + 90% S and 20% SCG + 80% Sand 30% S C G + 70% S and 100 % S CG P N N eedle 1 .09 (0.04) c 0 .86 (0.02) b 1 .30 (0.02) d 1 .52 (0.05) e 1 .45 (0.02) e 2 .65 (0.02) a 0 .000 Lateral root 1 .10 (0.02) c 1 .00 (0.02) b 1 .2 6 (0.02) d 1 .51 (0.03) e 1 .60 (0.04) a * 0.000 Main root 0 .63 (0.01) b 0 .65 (0.01) b 0 .82 (0.03) c 1 .04 (0.04) d 1 .05 (0.02) d 2 .80 (0.05) a 0 .000 Stem 0 .57 (0.03) b 0 .54 (0.01) b 0 .76 (0.01) c 1 .11 (0.06) d 1 .03 (0.04) d 2 .99 (0.01) a 0 .000 C N eedle 5 2.07 (0.38) a 48.91 (0.82) bc 50.80 (0.41) ac d 49.90 (0.55) bcd 51.46 (0.36) ad 48.67 (0.56) b 0 .000 Lateral root 4 5.17 (0.54) a 44.77 (0.38) a 41.85 (0.62) c 40.73 (0.64) bc 39.55 (0.97) b * 0.000 Main root 4 9 .93 (0.43) a 49.68 (0.24) a 50.68 (0.53) a 49.22 (0.48) a 50.09 (0. 55) a 49.38 (0.90) a 0 .340 Stem 5 0 .6 (0.40) a 51.07 (0.39) a 50.60 (0.45) a 49.35 (0.55) ab 51.04 (0. 61) a 48.42 (0.91) b 0 .036 Ca Needle 3 082.21 (72 .44) c 5137.44 (148.15) a 4548.7 9 (115.89) f 398 0.21 (105.38) e 3528.77 (107.29) d 252 2.00 (64.87) b 0 .000 Lateral root 8 136.46 (16 8 .11) c 14,987.18 (5 24.55) a 13, 389 .61 (291.08) e 10,108.9 4(238. 29) d 3516.60 (916.77) b * 0.000 Main root 2 467.44 (42 .90) c 4875.11 (136.77) a 3852.2 8 (34.63) d 362 5.75 (153.80) d 2823.70 (306.26) c 139 1.41 (46.73) b 0 .000 Stem 1 983.38 (48 .11) b 3186.33 (361.88) a 3157.0 3 (87.49) a 289 9.25 (63.09) a 2737.03 (85.93) a 156 9.85 (23.99) b 0 .000 K N eedle 5 592.13 (13 7.62) c 3272.00 (170.94) b 5782.8 7 (141.52) c 707 4.20 (225.15) a 7002.37 (123.08) a 683 9.77 (18.90) a 0 .000 Lateral root 4 513.24 (10 7 .13) a 2660.58 (84.29) b 3756.3 6 (100.46) c 459 7.85 (101.49) a 2303.51 (634.03) bc * 0 .000 Main root 5 439.88 (64 .69) ac 3834.39 (92.26) b 5178.0 5 (119.94) c 604 7.56 (158.42) a 4767.17 (478.50) c 303 6.31 (89.39) b 0 .000 Stem 5 393.50 (63 .28) a 2611.18 (308.82) b 4286.3 9 (98.44) c 458 6.44 (30.83) c 4420.52 (71.10) c 233 4.35 (37.40) b 0 .000 Mg Needle 1 505.32 (20 .89) c 1319.66 (29.03) b 1375.7 9 (17.68) b 136 5.83 (19.38) b 1360.12 (20.21) b 170 9.81 (40.21) a 0 .000 Lateral root 1 686.50 (44 .33) a 1383.78 (16.06) c 1591.6 7 (18.19) ac 150 0.69 (23.42) ac 696.9 1 (185.50) b * 0.000 Main root 9 28.70 (16.37) bc 795.51 (15.6 6 ) b 880.72 (10.97) bc 973 .48 (31.38) c 875.4 3 (81.87) bc 119 6.71 (7.63) a 0 .001 Stem 1 111.70 (33 .33) ac 673.84 (66.5 6) b 1045.3 1 (13.95) c 123 0.58 (43.47) a 1150.14 (25.56) ac 118 6.73 (18.53) ac 0.000 P N eedle 1 330.92 (47 .50) e 702.49 (36.0 7 ) b 951.98 (33.87) c 113 5.55 (36.58) d 1210.89 (26.04) de 172 5.27 (56.57) a 0 .000 Lateral root 1 697.82 (54 .23) a 786.53 (41.0 4 ) b 871. 95 (21.19) b 920 .13 (31.11) b 567.0 8 (156.63) b * 0.000 Main root 1 757.94 (53 .86) e 816.19 (49.6 9 ) b 967.37 (57.98) bc 119 9.00 (68.17) cd 1253.80 (129.42) d 327 0.38 (21.58) a 0 .000 Stem 1 957.73 (91 .20) f 610.80 (70.5 4 ) b 889.14 (52.59) c 130 1.95 (60.06) d 1568.96 (60.69) e 341 4.64 (48.11) a 0 .000 C/N N eedle 4 8.21 (1.58) e 56.93 (1.04) a 39.23 (0.52) d 33.13 (0.85) c 35.44 (0. 43) c 18.35 (0.29) b 0 .000 Lateral root 4 1.14 (0.69) e 44.81 (0.87) a 33.27 (0.47) d 26.99 (0.43) c 24.71 (0.23) b * 0.000 Main root 7 9 .43 (1.22) a 77.29 (1.93) a 62.89 (2.60) d 48.12 (1.96) c 47.87 (0. 92) c 17.64 (0.10) b 0 .000 Stem 9 0 .92 (4.23) a 94.47 (1.98) a 66.75 (1.50) d 46.41 (3.10) c 50.35 (2. 05) c 16.19 (0.33) b 0 .000 V al u es ar e the me an; C and N ar e % ; C a, K ,Mg, and P are mg kg − 1 ; numbers in parenthesis are standard error, an dv al u esi nt h e sa m e ro wf o ll o w edb yt h e sa m e le tt er are not statistically different at the 0.05 sig n ifican ce level in the Duncan post hoc test . * Lack of samples in 100% SCG for lateral root

4 Discussion

As shown by the results, GP started to decrease and MGT value increased in proportion to the increase of SCG in the media. In other words, the seeds germinated later and grew less with the increase of SCG in the growing media. The germination percentage did not show a decrease until grown in the media that contained 30% SCG + 70% sand. Media containing 10 and 20% SCG did not adversely affect germi-nation. Comments made for GP also apply to MGT. It can be said that the EC values of the media were effective in the decrease in germination percentage. Depending on the salini-zation of the media, it became difficult for the seeds to absorb water and develop roots. No study was encountered in the literature on the germination properties of tree seeds in media containing different amounts of coffee wastes.

However, total biomass in 1-year-old seedlings reduced in media where SCG was added, but some important plant pa-rameters were improved. For example, in 10% SCG + 90% sand, the root/shoot ratio reached the highest value, which was more than 50% of the root/shoot ratio in conventional media. Root/shoot ratio is a very important indicator for seed-ling quality. Root tissue represents support for nutrition and shoots represent growth. From this point of view, higher ratios can support root tissues, which in turn will increase the ab-sorption of soil nutrients, and more nutrition uptake will in-crease photosynthesis and carbon storage.

Additionally, compared with conventional media and 100% sand, the number of lateral branches increased with an increase in SCG ratio. However, the increase in the number of lateral branches did not lead to differentiation in total needle weights. Although no studies have been conducted with woody species in the literature, plant trials with SCG have been conducted often on agricultural products. In most of these studies, a decrease was observed in agricultural yield upon direct addition of SCG to the media. For example, the addition of SCG to the media reduced growth in lettuce by 233% (Cervera-Mata et al.2018). With soil pots containing coffee waste involving 12 different agricultural plants, high concentrations of coffee wastes slowed down plant growth (Kitou and Yoshida,1997as cited in Yamane et al.2014). Ribeiro et al. (2017) also stated that the application of SCG together with ash decreased growth in Lolium perenne. The addition of two different doses of SCG had a regressive effect on the development of lettuce, and growth parameters were negatively correlated with the SCG dose (Cervera-Mata et al. 2018). Hardgrove and Livesley (2016) stated that the devel-opment of broccoli, leek, radish, viola, and sunflower plants was poor in their experiment using SCG directly. However, some researchers have stated that the use of SCG with other additives gives beneficial results for plant growth. Kasongo et al. (2013), for instance, stated that the addition of SCG positively affected plant growth in highly acidic and sandy

(unfertile) soil conditions. Similarly, Ronga et al. (2016) re-ported that mixture of peat with up to 40% SCG increased plant growth and even gave as good results as regular fertilizers. Cruz et al. (2015) determined that in the case of 10% SCG + soil mixture, Batavia lettuce did not suffer any yield loss, but yield reduction was observed in mixtures con-taining 20% or 30% SCG. This suggests that high SCG rates are harmful for plants (McNutt 2019). The present study showed that the use of an SCG ratio of 30% and 100% had a negative effect on biomasses of different plant components. The direct addition of SCG to the media apparently retards growth in plants because caffeine, tannin, and polyphenol com-pounds contained in SCG have toxic effects on plants. Similar results have already been reported in the literature (Cervera-Mata et al. 2018; Ciesielczuk et al. 2018; Hardgrove and Livesley2016; Yamane et al.2014). However, in contrast to the above findings, Gomes et al. (2014) used coffee wastes in lettuce cultivation, tested coffee wastes in different concentra-tions as fresh (2.5; 5; 10; 15; 20%, v/v) and composted (5; 10; 15; 20; 30%, v/v), and stated that it had a positive effect on all growth parameters examined. In the present study as well, it was observed that 10% and 20% mixtures of SCG had a pos-itive effect on the growth of stone pine seedlings.

A very substantial factor in the waste utilization of SCG is the possible existence of toxic compounds for plants. Although it has not been tested and was not among the aims of the present study, evaluation on toxicity effects of SCG has been considered important and necessary. Caffeine, tannins, and polyphenols among the compounds in SCG are specified as toxic for plants; they are also defined as phytotoxic and eco-toxic (Mussatto et al.2011; Hachicha et al.2012; Kim et al. 2014; Ciesielczuk et al. 2017, 2018; Janissen and Huynh 2018; Vasmara and Marchetti2018).

Samples of toxicity effects were described as reduced prima-ry root, root hair length, the total number of root hairs (Janissen and Huynh2018), and significant reductions in seed germina-tions on some species such as Lepidium (Ciesielczuk et al. 2018) and Arabidopsis thaliana (Janissen and Huynh2018). On the other hand, Saadi et al. (2007) have not demonstrated the direct toxic effect of polyphenols on plants suggesting in-teraction with fatty acids (as described in Ciesielczuk et al. 2018). Also, Cruz et al. (2015) could not confirmed the toxicity of SCG in pot and field experiments for lettuce, and recom-mended the possibility for extensive use of SCG as fertilizer due to high levels of antioxidant capacity. There is strong evi-dence to suggest that SCG has great potential as a fertilizer; however, there is a major gap on its’ toxic effects (Janissen and Huynh2018). In addition, Hachicha et al. (2012) empha-sized that if composting of SCG increased C/N and toxicity levels decreased, C/N ratio decreased from the initial 21 to 14–16 and the polyphenol toxicity effect decreased by 65– 72% at the end of composting (about 15 weeks) in two different SCG mixtures. In our study, C/N ratio was 16 in pure SCG

media and C/N ratio decreased as SCG increased in the other medias likely indicating more composted SCG in the present study.

As described above, the main limitation of the present study that toxicity effects of SCG was not determined and evaluated, and it can be highly recommended for further research. If the results of Janissen and Huynh (2018) and Ciesielczuk et al. (2018) are considered, significantly low root biomass and germination in pure SCG media among our re-sults can be interpreted as possible toxicity impacts of SCG.

Despite the varied results in plant growth, the addition of SCG to growing media has soil-improving effects. Although not covered by the scope of this study, SCG as an organic amendment agent can improve the water retention and aeration conditions of soil by improving its physical properties. Cervera-Mata et al. (2019a) stated that the addition of SCG to two types of soil with high clay content in Spain increased the water holding capacity and the amount of available water to the plant, raised the pore volume three times, reduced the soil bulk den-sity, and improved soil physical properties through soil aggre-gates. With its high nutrient content, SCG also improves the chemical properties of soil. In this study, the addition of SCG at different rates caused a rise in N, C, K, Mg, and P in the growth media compared to in 100% sand. In fact, some research results (Mussatto et al.2011) indicate much higher nutrient concentra-tions than the present study. Cuevas and Quiroz (2019), indi-cated a minor proportion of the applied dairy slurry was absorbed by tested several tree species uptaking more P, Ca, Mg, and Na and responded positively on a dry matter basis. Similarly, Yamane et al. (2014) tested the use of coffee waste as a soil amendment agent and agricultural product enhancer in agricultural areas and found that it increased the carbon and nitrogen content in the soil and reduced the C/N ratio. Marzi et al. (2020) tested six types of organic amendments were ap-plied to three types of soils at the laboratory scale. Those or-ganic residues with high ratios of C/N immobilized the mineral nitrogen resulting in suppressed levels of nitrogen in the amended soil. Besides, Choo et al. (2020) described that lower rates of organic waste (pineapple residue ash) increased the nitrogen uptake by tested plant (Ananas comosus L. Merr.). Kondamudi et al. (2008) point out the high concentrations of N (1.2–2.3%), P (0.02–0.5%), and K (≈ 0.35%) with SCG and suggest its use as soil amendment agent and fertilizer. SCG contains ash and various minerals, potassium being the most abundant element, followed by phosphorus and magnesium (Mussatto et al.2011). Total mineral content (K, Mg, P, Ca, Na, Fe, Mn, and Cu) varies between 0.82 and 3.52%. Potassium was determined to be between 3.12 and 21.88 mg g−1(Cruz et al.2012). Magnesium makes up 11% of the total SCG minerals (Mussatto et al.2011). The highest concentration of SCG in the present study was magnesium with 2836 mg kg−1, followed by Ca with 2662 mg kg−1, P with 884 mg kg−1, and K with 630 mg kg−1. However, Ibeto et al.

(2020) concluded that wood ash treatments had lower uptake and transfer factors for the metals in carrot.

The high nutrient content of SCG added to media increases plant nutrient concentrations. In this study, it was observed that the nutrients (N, K, Mg, P) except Ca tended to increase in different plant components. As mentioned above, all SGC studies have been performed on the species used to obtain agricultural plants, thus it is not possible to make a compari-son with a woody species. But to give an example from the literature, Ribeiro et al. (2017) also emphasize that SCG ap-plication with Lolium perenne increases the chemical nutrients (available macronutrients Ca, Mg, K, and P). The addition of SCG to the media for lettuce cultivation increased total N and available K and P concentrations by 286%, 188%, and 9%, respectively (Cervera-Mata et al. 2018). Cervera-Mata et al. (2019b) found high concentrations of Fe, Mn, and Zn in let-tuces grown in agricultural soil by adding SCG directly, pointing out that although the toxic elements Al and As were high, they did not reach toxic limits. Thus, the addition of SCG allows plant nutrition with greater element concentra-tions. However, despite the high concentration of Ca in the SCG used in the present study, Ca ratios in stone pine com-ponents decreased as SCG increased. In other words, despite the presence of SCG with a high Ca content, Ca cannot be taken up by stone pine seedlings.

5 Conclusions

In the changing world, reuse of organic wastes is a strategy of great interest. In this perspective, the chemical composition of coffee wastes attracts attention to the various re-uses. The trials on the effects of adding the spent coffee grounds on plant growth involve species mostly used in agricultural purposes. There are no studies conducted with woody species. The pres-ent study is a first pioneer research on a forest tree species and the results clearly revealed that the use of low concentrations of spent coffee grounds has a positive effect on 1 + 0 stone pine seedlings. This can be interpreted that spent coffee grounds might be alternative to inorganic fertilizers for forest tree nurseries; however, further studies are needed on different tree species, processes, or test on field conditions for efficient-ly utilizing of coffee ground waste.

Acknowledgments The current study drew on the Postgraduate Thesis Study carried out by Nihan Özok under the consultancy of Assoc. Prof. Dr. Servet Çalışkan and Prof. Dr. Ender Makineci in the Institute of Graduate Studies, Forest Engineering, Istanbul University-Cerrahpasa. We thank Res. Asst. Safa Balekoglu for his valuable contributions for the collection of stone pine seed material. The authors thank to three anonymous reviewers and editor for valuable comments which are sig-nificantly improved the original manuscript. The authors thank to Moiz Cenkel for giving inspirations on reuse of coffee grounds.

Conflict of Interest The authors declare that they have no conflict of interest.

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